Objective Vascular cells, particularly endothelial cells, adopt aerobic glycolysis to generate energy to support cellular functions. The effect of endothelial glycolysis on angiogenesis remains unclear. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, isoform 3 (PFKFB3), is a critical enzyme for endothelial glycolysis. By blocking or deleting PFKFB3 in endothelial cells, we investigated the influence of endothelial glycolysis on angiogenesis both in vitro and in vivo. Approach and Results Under hypoxic conditions or following treatment with angiogenic factors, endothelial PFKFB3 was upregulated both in vitro and in vivo. The knockdown or overexpression of PFKFB3 suppressed or accelerated endothelial proliferation and migration in vitro, respectively. Neonatal mice from a model of oxygen-induced retinopathy showed suppressed neovascular growth in the retina when endothelial PFKFB3 was genetically deleted or when the mice were treated with a PFKFB3 inhibitor. Additionally, tumors implanted in mice deficient in endothelial PFKFB3 grew more slowly and were provided with less blood flow. A lower level of phosphorylated AKT (pAKT) was observed in PFKFB3-knockdown endothelial cells, which was accompanied by a decrease in intracellular lactate. The addition of lactate to PFKFB3-knockdown cells rescued the suppression of endothelial proliferation and migration. Conclusions The blockade or deletion of endothelial PFKFB3 decreases angiogenesis both in vitro and in vivo. Thus, PFKFB3 is a promising target for the reduction of endothelial glycolysis and its related pathological angiogenesis.

Non-alcoholic fatty liver disease (NAFLD) is closely associated with obesity and insulin resistance. To better understand the pathophysiology of obesity-associated NAFLD, the present study examined the involvement of liver and adipose tissues in metformin actions on reducing hepatic steatosis and inflammation during obesity. C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity-associated NAFLD and treated with metformin (150 mg/kg/d) orally for the last four weeks of HFD feeding. Compared with HFD-fed control mice, metformin-treated mice showed improvement in both glucose tolerance and insulin sensitivity. Also, metformin treatment caused a significant decrease in liver weight, but not adiposity. As indicated by histological changes, metformin treatment decreased hepatic steatosis, but not the size of adipocytes. In addition, metformin treatment caused an increase in the phosphorylation of liver AMP-activated protein kinase (AMPK), which was accompanied by an increase in the phosphorylation of liver acetyl-CoA carboxylase and decreases in the phosphorylation of liver c-Jun N-terminal kinase 1 (JNK1) and in the mRNA levels of lipogenic enzymes and proinflammatory cytokines. However, metformin treatment did not significantly alter adipose tissue AMPK phosphorylation and inflammatory responses. In cultured hepatocytes, metformin treatment increased AMPK phosphorylation and decreased fat deposition and inflammatory responses. Additionally, in bone marrow-derived macrophages, metformin treatment partially blunted the effects of lipopolysaccharide on inducing the phosphorylation of JNK1 and nuclear factor kappa B (NF-κB) p65 and on increasing the mRNA levels of proinflammatory cytokines. Taken together, these results suggest that metformin protects against obesity-associated NAFLD largely through direct effects on decreasing hepatocyte fat deposition and on inhibiting inflammatory responses in both hepatocytes and macrophages.

BACKGROUND AND PURPOSEImidazoline I2 receptors have been implicated in several CNS disorders. Although several I2 receptor agonists have been described, no simple and sensitive in vivo bioassay is available for studying I2 receptor ligands. This study examined I2 receptor agonist-induced hypothermia as a functional in vivo assay of I2 receptor agonism. EXPERIMENTAL APPROACHDifferent groups of rats were used to examine the effects of I2 receptor agonists on the rectal temperature and locomotion. The pharmacological mechanisms were investigated by combining I2 receptor ligands and different antagonists. KEY RESULTSAll the selective I2 receptor agonists examined (2-BFI, diphenyzoline, phenyzoline, CR4056, tracizoline, BU224 and S22687, 3.2-56 mg·kg -1 , i.p.) dose-dependently and markedly decreased the rectal temperature (hypothermia) in rats, with varied duration of action. Pharmacological mechanism of the observed hypothermia was studied by combining the I2 receptor agonists (2-BFI, BU224, tracizoline and diphenyzoline) with imidazoline I2 receptor/ a2 adrenoceptor antagonist idazoxan, selective I1 receptor antagonist efaroxan, a2 adrenoceptor antagonist/5-HT1A receptor agonist yohimbine. Idazoxan but not yohimbine or efaroxan attenuated the hypothermic effects of 2-BFI, BU224, tracizoline and diphenyzoline, supporting the I2 receptor mechanism. In contrast, both idazoxan and yohimbine attenuated hypothermia induced by the a2 adrenoceptor agonist clonidine. Among all the I2 receptor agonists studied, only S22687 markedly increased the locomotor activity in rats. CONCLUSIONS AND IMPLICATIONSImidazoline I2 receptor agonists can produce hypothermic effects, which are primarily mediated by I2 receptors. These data suggest that I2 receptor agonist-induced hypothermia is a simple and sensitive in vivo assay for studying I2 receptor ligands. Abbreviations

BackgroundPrevious studies propose that hypothyroidism might play a crucial role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), but findings from published studies on the relationship between hypothyroidism and NAFLD are still controversial. Our study aimed to make a comprehensive evaluation of the relationship between hypothyroidism and NAFLD through a meta-analysis.MethodsPubMed, China Dissertation Database, and EMBASE databases were searched to find observational studies assessing the relationship between hypothyroidism and NAFLD. The pooled odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated to evaluate the strength of the relationship between hypothyroidism and NAFLD through meta-analysis.ResultsThirteen articles were ultimately included in our meta-analysis. Meta-analysis of the 13 studies found a high correlation between hypothyroidism and NAFLD (OR = 1.52, 95% CI 1.24–1.87, P < 0.001). Meta-analysis of 9 studies providing adjusted ORs found that hypothyroidism was independently correlated with NAFLD (OR = 1.72, 95% CI 1.32–2.23, P < 0.001). Subgroup analysis found that both overt hypothyroidism and subclinical hypothyroidism were significantly correlated with NAFLD, and the pooled ORs were 1.70 (95% CI 1.23–2.36, P = 0.002) and 1.40 (95% CI 1.10–1.77, P = 0.006), respectively. Besides, meta-analysis of studies providing adjusted ORs also found that both overt hypothyroidism and subclinical hypothyroidism were independently correlated with NAFLD, and the pooled ORs were 1.81 (95% CI 1.30–2.52, P < 0.001) and 1.63 (95% CI 1.19–2.24, P = 0.002), respectively.ConclusionThe meta-analysis provides strong epidemiological evidence for the relationship between hypothyroidism and NAFLD. Both individuals with subclinical and overt hypothyroidism are at higher risk for NAFLD than euthyroid subjects.

Peptidylargininedeiminase 1 (PAD1) catalyzes protein for citrullination, and this activity has been linked to the epidermal cornification. However, a role for PAD1 in tumorigenesis, including breast cancers has not been previously explored. Here we first showed that PAD1 is overexpressed in human triple negative breast cancer (TNBC). In cultured cells and xenograft mouse models, PAD1 depletion or inhibition reduced cell proliferation, suppressed epithelial-mesenchymal transition, and prevented metastasis of MDA-MB-231 cells. These changes were correlated with a dramatic decrease in MMP2/9 expression. Furthermore, ERK1/2 and P38 MAPK signaling pathways are activated upon PAD1 silencing. Treatment with MEK1/2 inhibitor in PAD1 knockdown cells significantly recovered MMP2 expression, while inhibiting P38 activation only slightly elevated MMP9 levels. We then showed that PAD1 interacts with and citrullinates MEK1 thereby disrupting MEK1-catalyzed ERK1/2 phosphorylation, thus leading to the MMP2 overexpression. Collectively, our data indicate that PAD1 appears to promote tumorigenesis by regulating MEK1-ERK1/2-MMP2 signaling in TNBC. These results also raise the possibility that PAD1 may function as an important new biomarker for TNBC tumors and suggest that PAD1-specific inhibitors could potentially be utilized to treat metastatic breast cancer.